The present invention relates to the pulmonary administration of a therapeutic protein and, more particularly, to the systemic administration via the respiratory system of therapeutically effective amounts of granulocyte colony stimulating factor (G-CSF) or chemically modified G-CSF. In another aspect, the present invention relates to the pulmonary administration of a pegylated protein.
G-CSF is a hormone-like glycoprotein which regulates hematopoiesis and is required for the clonal growth and maturation of normal hematopoietic precursor cells found in the bone marrow; Welte et al., Proc. Natl. Acad. Sci., Vol. 82, pp. 1526-1530 (1985). More specifically, G-CSF, when present in low concentrations, is known to stimulate the production of neutrophil granulocytic colonies when used in vitro. G-CSF is also known to enhance neutrophil migration; Gabrilove, J., Seminars in Hematology, Vol. 26, No. 2, pp. 1-4 (1989). Moreover, G-CSF can significantly increase the ability of neutrophils to kill tumor cells in vitro through antibody mediated cellular cytotoxicity; Souza et al., Science, Vol. 232, pp. 61-65 (1986).
In humans, endogenous G-CSF is detectable in blood plasma; Jones et al., Bailliere""s Clinical Hematology, Vol. 2, No. 1, pp.83-111. G-CSF is produced by fibroblasts, macrophages, T cells, trophoblasts, endothelial cells and epithelial cells and is the expression product of a single copy gene comprised of four exons and five introns located on chromosome seventeen. Transcription of this locus produces a mRNA species which is differentially processed, resulting in the expression of two forms of G-CSF, one version having a mature length of 177 amino acids, the other having a mature length of 174 amino acids. The form comprised of 174 amino acids has been found to have the greatest specific in vivo biological activity. G-CSF is species cross-reactive, such that when human G-CSF is administered to another mammal such as a mouse, canine or monkey, sustained neutrophil leukocytosis is elicited; Moore et al., Proc. Natl. Acad. Sci., Vol. 84, pp. 7134-7138 (1987).
Human G-CSF can be obtained and purified from a number of sources. Natural human G-CSF (nhG-CSF) can be isolated from the supernatants of cultured human tumor cell lines. The development of recombinant DNA technology, see, for instance, U.S. Pat. No. 4,810,643 (Souza), incorporated herein by reference, has enabled the production of commercial scale quantities of G-CSF in glycosylated form as a product of eukaryotic host cell expression, and of G-CSF in non-glycosylated form as a product of prokaryotic host cell expression.
Chemically modified G-CSF may also be obtained in numerous ways. Chemical modification may provide additional advantages, such as increasing the stability and clearance time of the therapeutic protein. A review article describing protein modification and fusion proteins is Francis, Focus on Growth Factors 3: 4-10 (May 1992)(published by Mediscript, Mountview Court, Friern Barnet Lane, London N20 OLD, UK). For example, see EP 0 401 384, entitled, xe2x80x9cChemically Modified Granulocyte Colony Stimulating Factor,xe2x80x9d which describes materials and methods for preparing G-CSF to which polyethylene glycol molecules are attached. (Such modified G-CSF is referred to herein as xe2x80x9cpegylated G-CSFxe2x80x9d or xe2x80x9cPEG-G-CSF.xe2x80x9d). Such chemically modified G-CSF may be obtained by modifying nhG-CSF or G-CSF obtained as a product of prokaryotic or eukaryotic host cell expression.
G-CSF has been found to be useful in the treatment of cancer, as a means of stimulating neutrophil production to compensate for hematopoietic deficits resulting from chemotherapy or radiation therapy. The effective use of G-CSF as a therapeutic agent requires that patients be administered systemic doses of the protein. Currently, parenteral administration via intravenous, intramuscular or subcutaneous injection is the preferred route of administration to humans and has heretofore appeared to be the only practical way to deliver therapeutically significant amounts of G-CSF to the bloodstream, although attempts have been made at oral delivery; see, for example, Takada et al., Chem. Pharm. Bull., Vol. 37, No. 3, pp. 838-839 (1989). Pulmonary delivery of chemically modified G-CSF has not been demonstrated previously, nor has pulmonary delivery of protein to which one or more polyethylene glycol molecules has been attached.
The pulmonary delivery of relatively large molecules is not unknown, although there are only a few examples which have been quantitatively substantiated. Leuprolide acetate is a nonapeptide with luteinizing hormone releasing hormone (LHRH) agonist activity having low oral availability. Studies with animals indicate that inhalation of an aerosol formulation of leuprolide acetate results in meaningful levels in the blood; Adjei et al., Pharmaceutical Research, Vol. 7, No. 6, pp. 565-569 (1990); Adjei et al., International Journal of Pharmaceutics, Vol. 63, pp. 135-144 (1990).
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide produced by endothelial cells, has been found to decrease arterial blood pressure when administered by aerosol to guinea pigs; Braquet et al., Journal of Cardiovascular Pharmacology, Vol. 13, suppl. 5, s. 143-146 (1989).
The feasibility of delivering human plasma xcex11-antitrypsin to the pulmonary system using aerosol administration, with some of the drug gaining access to the systemic circulation, is reported by Hubbard et al., Annals of Internal Medicine, Vol. III, No. 3, pp. 206-212(1989)
Pulmonary administration of xcex1-1-proteinase inhibitor to dogs and sheep has been found to result in passage of some of that substance into the bloodstream; Smith et al., J. Clin. Invest., Vol. 84, pp. 1145-1146 (1989).
Experiments with test animals have shown that recombinant human growth hormone, when delivered by aerosol, is rapidly absorbed from the lung and produces faster growth comparable to that seen with subcutaneous injection; Oswein et al., xe2x80x9cAerosolization of Proteinsxe2x80x9d, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colo., March, 1990. Recombinant versions of the cytokines gamma interferon (IFN-xcex3) and tumor necrosis factor alpha (TNF-xcex1) have also been observed in the bloodstream after aerosol administration to the lung; Debs et al., The Journal of Immunology, Vol. 140, pp. 3482-3488 (1988).
Pulmonary administration of pegylated proteins has not been demonstrated previously, although, as noted above, chemical modification of proteins, including pegylation, has been demonstrated for a variety of proteins, including G-CSF.
The present invention is based on the discovery that G-CSF can be administered systemically to a mammal via the pulmonary route. Typically, this is accomplished by directing a stream of a therapeutically effective amount of G-CSF into the oral or nasal cavity of the inhaling mammal. Importantly, and surprisingly, substantial amounts of G-CSF are thereby deposited in the lung and absorbed from the lung into the bloodstream, resulting in elevated blood neutrophil levels. Moreover, this is accomplished without the necessity to resort to special measures such as the use of absorption enhancing agents or protein derivatives specifically designed to improve absorption. Pulmonary administration of G-CSF thus provides an effective non-invasive alternative to the systemic delivery of G-CSF by injection.
In another aspect, the present invention is based on the discovery that chemically modified G-CSF may be absorbed from the lung into the bloodstream. In addition to the advantages of pulmonary delivery as described above, this provides additional advantages. Chemical modification may lengthen the circulation time of the protein in the body, alter immunoreactivity, reduce toxicity, alter bioactivity, and alter certain physical properties of the therapeutic peptide.
In yet another aspect, the present invention is based on the broad discovery that a protein to which a polyethylene glycol molecule has been attached may be absorbed by the lung into the bloodstream. Polyethylene glycol, or xe2x80x9cPEGxe2x80x9d is a hydrophilic polymer. For example, solid PEGs are insoluble in liquid paraffin, fats and fixed oils. Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association and The Pharmaceutical Society of Great Britian), pages 209-213 at 210. Further, the structure of the lung is such that while gaseous exchange is facilitated, uptake of solids or liquids is not. The membrane junctions between the epithelia cells are considered tight, and as such, would not be expected to allow absorption or transfer of large hydrophilic molecules. As such, PEG is not expected to cross hydrophobic membranes to any significant degree.
The pulmonary administration of G-CSF or chemically modified G-CSF can be practiced using any purified isolated polypeptide having part or all of the primary structural conformation (i.e., continuous sequence of amino acid residues) and one or more of the biological properties of naturally occurring G-CSF. A number of publications describe methods of producing G-CSFs, including the above mentioned Souza patent and the Welte et al. and Nicola et al. articles.
In general, G-CSF useful in the practice of this invention may be a native form isolated pure from mammalian organisms or, alternatively, a product of chemical synthetic procedures or of procaryotic or eucaryotic host expression of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis. Suitable procaryotic hosts include various bacterial (e.g., E. coli) cells. Suitable eucaryotic hosts include yeast (e.g., S. cerevisiae) and mammalian (e.g., Chinese hamster ovary, monkey) cells. Depending upon the host employed, the G-CSF expression product may be glycosylated with mammalian or other eucaryotic carbohydrates, or it may be non-glycosylated. The G-CSF expression product may also include an initial methionine amino acid residue (at position xe2x88x921). The present invention contemplates the use of any and all such forms of G-CSF, although recombinant G-CSF, especially E. coli derived, is preferred for reasons of greatest commercial practicality.
The G-CSF to be chemically modified for use in the present invention may also be either nhG-CSF or the product of a recombinant nucleic acid process, such as prokaryotic or eukaryotic host cell expression. In general, chemical modification contemplated is the attachment of a chemical moiety to the G-CSF molecule itself, where said chemical moiety permits pulmonary administration of the chemically modified G-CSF. The attachment may be by bonding, directly to the protein or to a moiety which acts as a bridge to the active agent. Covalent bonding is preferred as the most stable for attachment. The chemical modification may contribute to the controlled, sustained or extended effect of the G-CSF. This may have the effect, for example, of controlling the amount of time the chemically modified G-CSF takes to reach the circulation. An example of a chemical modifier is polyethylene glycols, including derivatives thereof.
Contemplated for use in the practice of this invention are any chemically modified G-CSF preparations which permit efficacy upon pulmonary administration. Efficacy may be determined by known methods, as a practitioner in the art will recognize. Pegylated G-CSF, especially pegylated E. coli derived G-CSF, and more particularly, tri-tetra pegylated E. coli derived G-CSF (as described below) is preferred.
When attaching the chemical moiety to the G-CSF or other peptide, one should consider the location of the attachment. For example, attachment in a location affecting the receptor binding site, functional domains or antigenic domains may also affect the biological activity.
Contemplated for use in the practice of pulmonary administration of a pegylated protein are a variety of compositions for which pulmonary administration would be desired in pegylated form. Exemplary proteins contemplated are cytokines, including various hematopoietic factors such as G-CSF, SCF, EPO, GM-CSF, CSF-1, the interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12, IGFs, M-CSF, thymosin, TNF, or LIF. Other therapeutic proteins such as interferons (alpha-, beta-, gamma- or consensus interferons) and growth factors or hormones are also useful, such as human or other animal growth hormones (for example, bovine, porcine, or chicken growth hormone), insulin, ET-1, FGF, KGF, EGF, IGF, PDGF, and xcex1-1 antitrypsin. Protease inhibitors, such as metalloproteinase inhibitors are contemplated (such as TIMP-1, TIMP-2, or other proteinase inhibitors). Nerve growth factors are also contemplated, such as BDNF and NT3. Plasminogen activators, such as tPA, urokinase and streptokinase are also contemplated. Also contemplated are peptide portions of proteins having all or part of the primary structural conformation of the parent protein and at least one of the biological properties of the parent protein. Analogs, such as substitution or deletion analogs, or those containing altered amino acids, such as peptidomimetics are also contemplated.
Also contemplated is use of polyethylene glycol molecules with a range of molecular weights. Preferred are those polyethylene glycol molecules which act to increase the half life of the peptide, typically those PEG molecules with a molecular weight of between about 500 and about 20,000. The term xe2x80x9caboutxe2x80x9d is used to reflect the approximate average molecular weight of a polyethylene glycol preparation, recognizing that some molecules in the preparation will weigh more, some less. Preferred are xe2x80x9csolidxe2x80x9d PEGs which are insoluble in fats and oils. xe2x80x9cSolidxe2x80x9d PEGs are generally of MW 1000 or above, although PEG 600 can be solid at ambient temperatures. Handbook of Pharmaceutical Excipients, supra, page 209, which is incorporated by reference. The PEG used in the working examples described below had a molecular weight of about 6000, and acted to increase the half life of the G-CSF used.
The polyethylene glycol molecules should be attached to the peptide with consideration of effects on functional or antigenic domains as noted above. The method for attachment of the polyethylene glycol molecules may vary, and there are a number of methods available to those skilled in the art. E.g., EP 0 401 384 (coupling PEG to G-CSF), see also, Malik et al, Exp. Hematol. 20: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydrl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
The number of polyethylene glycol molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function. As noted in more detail below, the pegylated G-CSF preferred herein is tri-tetra pegylated with PEG 6000, i.e., a G-CSF molecule having three or four PEG 6000 molecules attached.
Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass.
All such devices require the use of formulations suitable for the dispensing of G-CSF. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in G-CSF therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified G-CSF may also be prepared in different formulations depending on the type of chemical modification or the type of device employed. G-CSF formulations which can be utilized in the most common types of pulmonary dispensing devices to practice this invention are now described, and the same factors should be taken into consideration when formulating chemically modified G-CSF or a pegylated protein for pulmonary administration.
Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise G-CSF (or chemically modified G-CSF or pegylated protein) dissolved in water at a concentration of about 0.1 to 25 mg of G-CSF (or chemically modified G-CSF or pegylated protein) per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). Examples of buffers which may be used are sodium acetate, citrate and glycine. Preferably, for G-CSF formulations, the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of 3 to 4. Generally, buffer molarities of from 2 mM to 50 mM are suitable for this purpose. Examples of sugars which can be utilized are mannitol and sorbitol, usually in amounts ranging from 1% to 10% by weight of the formulation.
The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation. An especially preferred surfactant for purposes of this invention is polyoxyethylene sorbitan monooleate.
Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing G-CSF (or chemically modified G-CSF or a pegylated protein) suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing G-CSF (or chemically modified G-CSF or a pegylated protein) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The G-CSF (or chemically modified G-CSF or pegylated protein) should most advantageously be prepared in particulate form with an average particle size of less than 10 xcexcm (or microns), most preferably 0.5 to 5 xcexcm, for most effective delivery to the distal lung.
The invention contemplates the administration of therapeutic amounts of the protein, i.e., G-CSF or chemically modified G-CSF, sufficient to achieve elevation of the neutrophil level in the systemic blood. What constitutes a therapeutically effective amount of G-CSF or chemically modified G-CSF in a particular case will depend on a variety of factors which the knowledgeable practitioner will take into account, including the normal blood neutrophil level for that subject, the severity of the condition or illness being treated, the degree of neutropenia, the physical condition of the subject, and so forth. In general, a dosage regimen will be followed such that the normal blood neutrophil level for the individual undergoing treatment is restored, at least in cases of abnormally low or depressed blood neutrophil counts. For humans, the normal blood neutrophil level is about 5000 to 6000 neutrophils per microliter of blood. Neutrophil counts below 1000 in humans are generally regarded as indicative of severe neutropenia and as placing the subject at great risk to infection. Clinical studies with cancer patients suffering from chemotherapy-induced neutropenia have shown that subcutaneous injected doses of 3-5 xcexcg G-CSF/kg every twenty-four hours are effective in elevating acutely deficient blood neutrophil levels above 1000. Based on preliminary results with animals, described below, it is anticipated that for most mammals, including humans, the administered dose of G-CSF for pulmonary delivery (referred to here as the inhalation dose) will be about 3 to 10 times the corresponding subcutaneous dose necessary to achieve a particular blood neutrophil level.
The therapeutic dosage for chemically modified G-CSF may be ascertained taking into account the variety of factors listed above. Some chemical modification, such as pegylation, may lengthen the half-life of G-CSF in the body, and this should also be considered when ascertaining therapeutic dosage. Chemical modification may also alter immunoreactivity, reduce toxicity, alter bioactivity, and alter certain physical properties of the therapeutic protein, additional variables to consider when ascertaining therapeutic dosage of chemically modified G-CSF. Pegylated G-CSF, the chemically modified G-CSF used herein as an example, is known to have a serum half-life greater than that of non-pegylated G-CSF.
For other pegylated proteins, one skilled in the art should consider that pegylation may modify the pharmacological properties of proteins, usually extending the plasma half-life and concomitantly increasing in vivo bioactivity, and may reduce antigenicity and immunogenicity. There may also be an increase in solubility and resistance to proteolysis. For other general considerations when determining dosages, see Remington""s Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co., Easton, Pa.) Chapter 35.
As those skilled in the art will recognize, the operating conditions for delivery of a suitable inhalation dose will vary according to the type of mechanical device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and operating period will be dictated chiefly by the amount of G-CSF or other active composition per unit volume in the aerosol. In general, the higher the concentration of protein in the nebulizer solution the shorter the operating period. Some devices such as metered dose inhalers may produce higher aerosol concentrations than others and thus will be operated for shorter periods to give the desired result.
Other devices such as powder inhalers are designed to be used until a given charge of active material is exhausted from the device. The charge loaded into the device will be formulated accordingly to contain the proper inhalation dose amount of G-CSF or other active ingredient for delivery in a single administration. See generally, Remington""s Pharmaceutical Sciences, (18th Ed. 1990, Mack Publishing Co., Easton, Pa.) Chapter 92 for information relating to aerosol administration.
Regardless of what device is used for aerosolization, the active ingredients will be in the form of a dispersion of particles. The dispersion of particles may be in the form of liquid droplets or in the form of powder (dry or suspended). The dispersant itself, including the G-CSF, the chemically modified G-CSF, or a pegylated protein, is the population of particles which is emitted from the delivery device for deposition within the lung. As set forth above, an average particle size (mass median diameter) of less than 10 xcexcm (or microns), most preferably 0.5 to 5 xcexcm, is used for most effective delivery to the distal lung. For example, the dispersion of particles may consist essentially of pegylated protein, such as the pegylated G-CSF described herein, in a pharmaceutically acceptable carrier.
While G-CSF has been found useful in treating neutrophil-deficient conditions such as chemotherapy related neutropenia, G-CSF is expected to also be effective in combating infections and in treating other conditions or illnesses where blood neutrophil levels elevated above the norm can result in medical benefit. As further studies are conducted, information will emerge regarding appropriate dosage levels for the administration of G-CSF in these latter cases. Also, as further studies are accomplished using pegylated G-CSF, it is expected that appropriate formulations and dosages will be ascertained for the same uses. It is expected that the present invention will be applicable as a non-invasive alternative in most instances where G-CSF (or chemically modified G-CSF or a pegylated proteins) is administered therapeutically by injection.